Maturation of Innate Responses to Mycobacteria over the First 9 Months of Life.

To determine the evolution of innate responses to mycobacteria early in life, whole blood or PBMC from newborns, as well as 10- and 36-wk-old infants, was incubated with BCG or TLR ligands. Innate cell expression of cytokines and maturation markers was assessed, as well as activation of the proinflammatory NF-kB– and MAPK-signaling pathways. Maturation of innate proinflammatory responses during the first 9 months of life may underlie more effective control of mycobacteria and other pathogens observed later in infancy, and age-related differential induction of Th1 responses by vaccination.

Absolute cell counts are typically measured in fresh samples, but this is impractical in large field studies. We compared quantification of leukocyte proportions and absolute counts with a novel assay that allows long-term cryopreservation of fixed leukocytes for later counting with routine real-time measurement as reference. Our novel method allows accurate flow cytometric quantification of cell subsets from fixed WB even after long-term cryopreservation. This method is ideal for batched, retrospective analysis of samples from large field studies, or when advanced flow cytometry equipment is not available for clinical research purposes.

.We measured expression of total IFN-g from cellular subsets of whole blood, in response to stimulation with ESAT-6/CFP-10 peptide pools, in M.tb-infected and unifected participants, by inctracellular cytokine staining and flow cytometry. Remarkably, the primary source of IFN-g in the QuantiFERON assay has until now not been definitive. Our results show that CD4 T cells are the principal source of IFN-g in response to ESAT-6/CFP-10.

We aimed to qualify SATVI’s 12hr whole blood intracellular cytokine assay and assess the effects of long-term cryopreservation on outcomes. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity through measurement of mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17. We determined that the whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Kagina et al, J. Imm, Methods, 2014

Longitudinal characterisation of the HIV-1 specific T-lymphocyte immune responses over the first year of life in HIV-1 infected infants.

Whole blood samples from BCG-vaccinated HIV-infected infants were enrolled at 3, 6, 9 and 12 months of age and blood was collected at each time point. Blood was stimulated with HIV antigens Gag and Env, and antigen-specific cytokine production and expression of immunoregulatory molecules associated with HIV disease progression measured. We are currently analyzing the data.

To identify BCG-induced immune correlates of risk of tuberculosis, blood from 5,675 infants, who were routinely vaccinated with BCG at birth, was collected, processed and cryopreserved at 10 weeks of age. The infants were followed for at least 2 years to identify those that developed tuberculosis. We are using 2 approaches to find markers that correlate with risk of developing tuberculosis disease: hypothesis-driven and hypothesis-generating (global screening) approaches. Results of the first aim, to investigate whether the frequency or quality of BCG-specific T cell responses correlate with risk of tuberculosis have been published.

This study is aimed at determining whether innate genetic factors are associated with risk of developing childhood tuberculosis. DNA is being isolated to study polymorphisms of innate immunity genes, such as TLRs and NDRs, in the 2 groups.

Randhawa et al., PLoS Pathogens, 2011

Shey et al., Genes and Immunity, 2010

A study of the kinetics of the immune response induced by BCG vaccination of newborns.

This study is critical for determining when a new TB vaccine, aimed at boosting the BCG-induced response, might be administered to infants. Infants routinely vaccinated with BCG at birth were enrolled. From each infant, blood was collected at 3 of 7 time points over the first year of life. 33 healthy infants were enrolled per time point and BCG-specific T cell cytokine expression, proliferation and cytotoxic capacity were measured. We are currently completing data analysis.

This study aimed to characterisation T cells induced by MVA85A vaccination directly ex vivo, using HLA class II tetramers. We measured the memory phenotype, activation as well and integrin expression of MVA85A-induced T cells up to 1 year after vaccination.

Dintwe et al., Eur. J. Immunol, 2013

Delaying BCG vaccination from birth to 10 weeks of age may result in an enhanced memory CD4 T cell response.

To determine whether delaying BCG vaccination from birth to 10 weeks of age would result in an enhanced immune response to the vaccine.

Kagina et al, J Vaccine 2009

The role of Toll-Like Receptor 6 polymorphisms in innate immunity to Tuberculosis.

To determine if genetic variations in the TLR6 gene are associated with immunity to M. tuberculosis. The frequencies of TLR6 gene variants (polymorphisms) were determined by DNA sequencing. Blood collected from healthy adult participants from Cape Town was immediately incubated with M. tuberculosis extracts and TLR ligands and innate immune responses measured. We identified TLR6 gene variants that were associated with lower TLR6-mediated immune responses to TLR6 ligands.

Shey et al., Genes and Immunity, 2010

Development of a whole blood test to measure immunity caused by vaccines.

First, whole blood is incubated immediately after collection with specific antigens for 6-18 hours, ending with cryopreservation of plasma, fixed white cells, or RNA. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting; e.g., portable incubators that plug into car power sources are used to transport the incubated cells to the lab. Later, cryopreserved specimens may be thawed to measure cytokine producing CD4+ or CD8+ T cells in batch by flow cytometry, or to analyse plasma or RNA.

Proliferative responses, cytokine production and cell-mediated cytotoxicity obtained in post-vaccinated children were compared to baseline cord bloods and unvaccinated 10-week-old infants.

Hussey et al, J Immunol, 2002

The distal gut tract micobiome and vaccine immune responses in young African children.

To study the relationship between gut microbiome composition and vaccine-induced immune responses. Collection of stool samples from nappies of infants and young children enrolled in ongoing vaccine trials conducted at SATVI to generate a biobank for pilot studies.

O

Delayed BCG vaccination to study mechanisms of susceptability of infants to mycobacterial infection

To study a cohort of new-born infants where BCG vaccination is delayed from the immediate newborn period to 10 weeks of age, allowing comparison of host responses in unvaccinated babies with those in babies who receive BCG at birth.